1-[[1-[(2-amino-6-methyl-4-pyridinyl)methyl]-4-fluoro-4-piperidinyl]carbonyl]-4-[2-(2-pyridinyl)-3H-imidazo[4,5-B]pyridin-3-YL]piperidine and methods of use thereof

ABSTRACT

The present invention discloses the compound of Formula I 
                         
and pharmaceutically acceptable salts and solvates thereof. The invention also relates to pharmaceutical compositions comprising the Compound of Formula I and its use in treating obesity, metabolic syndrome, diabetes, hepatic lipidosis or nonalcoholic fatty liver disease. The invention also relates to the use of a combination of the Compound of Formula I with additional therapeutic agents for treating obesity, metabolic syndrome, diabetes, hepatic lipidosis or nonalcoholic fatty liver disease.

RELATED APPLICATIONS

This application is a division of U.S. patent. application Ser. No.11/523,489, filed Sep. 19, 2006, now allowed and herein incorporated byreference, which in turn claims benefit under 35 USC 119(e) to U.S.Provisional Patent Application No. 60/718,673, filed Sep. 20, 2005.

FIELD OF THE INVENTION

The present invention relates to1-[[1-[(2-amino-6-methyl-4-pyridinyl)methyl]-4-fluoro-4-piperidinyl]carbonyl]-4-[2-(2-pyridinyl)-3H-imidazo[4,5-b]pyridin-3-yl]piperidine,a compound useful as a histamine H₃ antagonist. The invention alsorelates to pharmaceutical compositions comprising said compound and itsuse in treating obesity, metabolic syndrome, diabetes, hepatic lipidosisor nonalcoholic fatty liver disease. The invention also relates to theuse of a combination of the histamine H₃ antagonist of this inventionwith other actives useful for treating obesity, metabolic syndrome,diabetes, hepatic lipidosis or nonalcoholic fatty liver disease. The useof the pharmaceutical compositions comprising the compound of theinvention with one or more compounds for treating obesity, metabolicsyndrome, diabetes, hepatic lipidosis or nonalcoholic fatty liverdisease are also contemplated.

BACKGROUND OF THE INVENTION

The histamine receptors, H₁, H₂, H₃ and H₄ have been characterized bytheir pharmacological behavior. The H₁ receptors are those that mediatethe response antagonized by conventional antihistamines. H₁ receptorsare present, for example, in the ileum, the skin, and the bronchialsmooth muscle of humans and other mammals. The most prominent H₂receptor-mediated responses are the secretion of gastric acid in mammalsand the chronotropic effect in isolated mammalian atria. H₄ receptorsare expressed primarily on eosinophils and mast cells and have beenshown to be involved in the chemotaxis of both cell types.

In the periphery, H₃ receptor sites are found on sympathetic nerves,where they modulate sympathetic neurotransmission and attenuate avariety of end organ responses under control of the sympathetic nervoussystem. Specifically, H₃ receptor activation by histamine attenuatesnorepinephrine outflow to resistance and capacitance vessels, causingvasodilation. In addition, in rodents, peripheral H₃ receptors areexpressed in brown adipose tissue, suggesting that they may be involvedin thermogenesis regulation.

H₃ receptors are also present in the CNS. H₃ receptor expression isobserved in cerebral cortex, hippocampal formation, hypothalamus andother parts of the human and animal brain. H₃ receptors are expressed onhistaminergic neurons and, as heteroreceptors, on neurons involved inother neurotransmitter systems, where H₃ receptor activation results inpresynaptic inhibition of neurotansmitter release. In the particularcase of histaminergic neurons, H₃ receptors have been implicated in theregulation of histamine hypothalamic tone, which in turn has beenassociated with the modulation of sleeping, feeding and cognitiveprocesses in the human brain (see, for example, Leurs et al., NatureReviews, Drug Discovery, 4, (2005), 107).

It is also known and has been described in the literature that histamineis involved in regulation of cognitive and memory processes in the humanbrain (see, for example, Life Sciences, 72, (2002), 409-414).Consequently, indirect modulation of histaminergic brain functionthrough the central H₃ receptors may be a means to modulate theseprocesses. Different classes of H₃ receptor ligands have been describedand their use for neurological and psychiatric diseases has beensuggested (see, e.g., US 20040224953, WO2004089373, WO2004101546). H₃receptor antagonists may be useful in treating various neuropsychiatricconditions, where cognitive deficits are an integral part of thedisease, specifically ADHD, schizophrenia and Alzheimer's disease (see,for example, Hancock, A.; Fox, G. in Drug Therapy (ed. Buccafusco, J.J.). (Birkhauser, Basel, 2003).

Imidazole H₃ receptor antagonists are well known in the art. Morerecently, non-imidazole H₃ receptor antagonists have been disclosed inU.S. Pat. Nos. 6,720,328 and 6,849,621, and in US Published Applications2004/0097483, 2004/0048843 and 2004/0019099. The present invention is aselection invention over US 2004/0097483.

SUMMARY OF THE INVENTION

The present invention provides the compound of formula I

and pharmaceutically acceptable salts and solvates thereof.

The invention also provides a pharmaceutical composition comprising aneffective amount of the compound of formula I and a pharmaceuticallyacceptable carrier.

The invention further provides methods for treating obesity, metabolicsyndrome, diabetes, hepatic lipidosis or nonalcoholic fatty liverdisease (each being a “Condition”) comprising administering to a patientin need of such treatment an effective amount of a Compound of FormulaI.

The invention further provides a pharmaceutical composition comprisingan effective amount of a combination of the Compound of Formula I, atleast one additional therapeutic agent, and a pharmaceuticallyacceptable carrier.

The invention further provides methods for treating a Conditioncomprising administering to a patient in need of such treatment: (i) aneffective amount of the Compound of Formula I and (ii) at least oneother additional therapeutic agent.

The invention also provides kits comprising a Compound of Formula I in apharmaceutical composition, and one or more additional therapeuticagents in separate pharmaceutical compositions, wherein all of theseparate pharmaceutical compositions are present in a single package.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the effect of the Compound of Formula I on triglyceridelevels in obese mice. The black bar on the left represents control mice(i.e., vehicle-treated mice) and the grey bar on the right representsmice treated with the Compound of Formula I (10 mg/kg/day for 12 daysadministered via gavage). The y-axis represents liver triglyceridelevels in mg/liver.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

“Effective amount” or “therapeutically effective amount” is meant todescribe an amount of compound or a composition of the present inventioneffective in inhibiting the above-noted diseases and thus producing thedesired therapeutic, ameliorative, inhibitory or preventative effect.

“Patient” means a human or non-human mammal. In one embodiment a patientis a human. In another embodiment, a patient is a non-human mammal. Inyet another embodiment, a patient is a companion animal, including butnot limited to, a dog, cat, rabbit, ferret or horse. In a specificembodiment, a patient is a dog. In another specific embodiment, apatient is a cat.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

Solvates of the Compound of Formula I are also contemplated herein.“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association mayinvolve varying degrees of ionic and covalent bonding, includinghydrogen bonding. In certain instances the solvate will be capable ofisolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolatable solvates. Non-limitingexamples of suitable solvates include ethanolates, methanolates, and thelike. “Hydrate” is a solvate wherein the solvent molecule is H₂O.Preparation of solvates is generally known. Thus, for example, M. Cairaet al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe thepreparation of the solvates of the antifungal fluconazole in ethylacetate as well as from water. Similar preparations of solvates,hemisolvate, hydrates and the like are described by E. C. van Tonder etal, AAPS Pharm Sci Tech., 5(1), article 12 (2004); and A. L. Bingham etal, Chem. Commun., 603-604 (2001). A typical, non-limiting, processinvolves dissolving the inventive compound in desired amounts of thedesired solvent (organic or water or mixtures thereof) at a higher thanambient temperature, and cooling the solution at a rate sufficient toform crystals which are then isolated by standard methods. Analyticaltechniques such as, for example I. R. spectroscopy, show the presence ofthe solvent (or water) in the crystals as a solvate (or hydrate).

The Compound of Formula I can form salts which are also within the scopeof this invention. Reference to the Compound of Formula I herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic saltsformed with inorganic and/or organic acids with some or all of the basicmoieties of the Compound of formula I, such as pyridine, benzimidazole,piperidine and aminopyridine. In one embodiment, a salt of the Compoundof Formula I is a Pharmaceutically Acceptable Salt (i.e., non-toxic,physiologically acceptable), Salts of the Compound of the Formula I maybe formed, for example, by reacting the Compound of Formula I with anamount of acid, such as an equivalent amount, in a medium such as one inwhich the salt precipitates or in an aqueous medium followed bylyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, fumarates, hydrochlorides,hydrobromides, hydroiodides, lactates, maleates, methanesulfonates,naphthalenesulfonates, nitrates, oxalates, phosphates, propionates,salicylates, succinates, sulfates, tartarates, thiocyanates,toluenesulfonates (also known as tosylates,) and the like. Additionally,acids which are generally considered suitable for the formation ofpharmaceutically useful salts from basic pharmaceutical compounds arediscussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook ofPharmaceutical Salts. Properties, Selection and Use. (2002) Zurich:Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977)66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33201-217; Anderson et al, The Practice of Medicinal Chemistry (1996),Academic Press, New York; and in The Orange Book (Food & DrugAdministration, Washington, D.C. on their website). These disclosuresare incorporated herein by reference thereto.

All such salts are intended to be pharmaceutically acceptable saltswithin the scope of the invention and all acid and base salts areconsidered equivalent to the free forms of the corresponding compoundsfor purposes of the invention.

The Compound of Formula I, and salts and solvates thereof, may exist intheir tautomeric form (for example, as an amide or imino ether). Allsuch tautomeric forms are contemplated herein as part of the presentinvention.

All stereoisomers (for example, geometric isomers, optical isomers andthe like) of the Compound of Formula I (including those of the salts,solvates and prodrugs of the compound as well as the salts and solvatesof the prodrugs), such as enantiomeric forms (which may exist even inthe absence of asymmetric carbons), rotameric forms, atropisomers, anddiastercomeric forms, are contemplated within the scope of thisinvention, individual stereoisomers of the Compound of Formula I may,for example, be substantially free of other isomers, or may be admixed,for example, as racemates or with all other, or other selected,stereoisomers. The use of the terms “salt”, “solvate”, “prodrug” and thelike, is intended to equally apply to the salt, solvate and prodrug ofenantiomers, stereoisomers, rotamers, tautomers, racemates or prodrugsof the inventive compound.

Polymorphic forms of the Compound of Formula I, and of the salts andsolvates of the Compound of Formula I, are intended to be included inthe present invention.

The term “metabolic syndrome” refers to a combination of risk factorsfor cardiovascular disease (CVD) identified in the National CholesterolEducation Program's Adult Treatment Panel III report. See for examplethe discussion by Grundy et al in Circulation, 109 (2004), 433-438. Thecomponents of metabolic syndrome are: 1) abdominal obesity; 2)atherogenic dyslipidemia; 3) raised blood pressure; 4) insulinresistance; 5) proinflamatory state; and 6) prothrombotic state.

The term “nonalcoholic fatty liver disease” or “NAFLD” describes aspectrum of liver diseases ranging from simple fatty liver (steatosis)to nonalcoholic steatohepatitis (ASH) with progressive fibrosis andliver failure. Hyperglycemia with or without evidence of hyperlipidemiais commonly associated with NAFLD. The disease exhibits the histologicalfeatures of alcohol-induced liver disease in patients who do not consumesignificant amounts of alcohol. All of the stages of NAFLD have incommon the accumulation of fat in the liver cells. Farrell and Larter inHepatology, 243: S99-S112 (2006) describe NASH as “the lynchpin” betweenhepatic steatosis and cirrhosis in the spectrum of NAFLD. See also,Palekar, et al., Liver Int., 26(2):151-6 (2006). In NASH, the fataccumulation of associated with varying degrees of inflammation andfibrosis. Conditions most commonly associated with NAFLD are obesity,type II diabetes and metabolic syndrome.

The terms “combination therapy” or “therapeutic combination” means theadministration of the Compound of Formula I and one or more additionaltherapeutic agents useful for treating a Condition. The combinations andtreatments of the present invention can be administered by any suitablemeans which produce contact of these compounds with the site of actionin the body, for example in the plasma, liver or small intestine of asubject (mammal or human or other animal). Such administration includescoadministration of these therapeutic agents in a substantiallysimultaneous manner such as in a single tablet or capsule having a fixedratio of active ingredients or in multiple, separate capsules for eachtherapeutic agent. Also, such administration includes use of each typeof therapeutic agent in a sequential manner. In either case, thetreatment using the combination therapy will provide beneficial effectsin treating the condition. A potential advantage of the combinationtherapy disclosed herein may be a reduction in the required amount of anindividual therapeutic compound or the overall total amount oftherapeutic compounds that are effective in treating the condition. Byusing a combination of therapeutic agents, the side effects of theindividual compounds can be reduced as compared to a monotherapy, whichcan improve patient compliance. Also, therapeutic agents can be selectedto provide a broader range of complimentary effects or complimentarymodes of action. The agents administered as combination therapy can actadditively or synergistically. In one embodiment, the combinationtherapies of the invention comprise the administration of the Compoundof Formula I and one additional therapeutic agent. In anotherembodiment, the combination therapies of the invention comprise theadministration of the Compound of Formula I and two additionaltherapeutic agents.

As used herein, the term “cholesterol lowering agent” means any compoundcapable of lowering the cholesterol level in patient.

The term “H₃ receptor antagonist/inverse agonist” refers to any compoundthat acts as an antagonist or an inverse agonist to an H₃ receptor in apatient.

The term “weight loss agent” refers to any compound capable of causing adecrease in the weight of a patient.

As used herein, “sterol absorption inhibitor” means a compound capableof inhibiting the absorption of one or more sterols, including but notlimited to cholesterol, phytosterols (such as sitosterol, campesterol,stigmasterol and avenosterol), 5α-stanols (such as cholestanol,5α-campestanol, 5α-sitostanol), and/or mixtures thereof, whenadministered in a therapeutically effective (sterol and/or 5α-stanolabsorption inhibiting) amount to a mammal or human.

The following abbreviations have the stated meanings: BOC istert-butoxycarbonyl; Ac is acetyl; Bu is butyl; conc. is concentrated;DMF is N,N-dimethylformamide; EDCI is1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; Et isethyl; EtOAc is ethyl acetate; EtOH is ethanol; HOBt ishydroxybenzotriazole; HPLC is high performance liquid chromatography; Meis methyl; MeOH is methanol; NaBH(OAc)₃ is sodium triacetoxyborohydride;Pr is propyl; i-PrOH is isopropanol; TFA is trifluoroacetic acid; andTHF is tetrahydrofuran.

Uses of the Compound of Formula I

The Compound of Formula I and pharmaceutically acceptable salts andsolvates thereof are useful for treating a Condition in a patient.

Accordingly, in one embodiment, the invention provides a method fortreating a Condition in a patient, comprising administering to thepatient an effective amount of the Compound of Formula I.

In one embodiment, the Condition being treated is obesity.

In another embodiment, the Condition being treated is metabolicsyndrome.

In another embodiment, the Condition being treated is nonalcoholic fattyliver disease.

In still another embodiment, the Condition being treated is diabetes. Inone embodiment, the diabetes is Type I diabetes. In another embodiment,the diabetes is Type II diabetes.

In a further embodiment, the Condition being treated is hepaticlipidosis.

Other Therapeutic Agents

The combination therapies of the present invention comprise theadministration of the Compound of Formula I and one or more additionaltherapeutic agents useful for treating a Condition.

Accordingly, in one embodiment, the invention provides a method for fortreating a Condition in a patient, comprising administering to thepatient: (i) an effective amount of the Compound of Formula I; and (ii)one or more additional therapeutic agents.

Additional therapeutic agents useful in the present methods include, butare not limited to cholesterol lowering agents, weight loss agents,antidiabetic agents and H₃ receptor antagonist/inverse agonists.

In one embodiment, the additional therapeutic agent is acholesterol-lowering agent.

In another embodiment, the additional therapeutic agent is a weight lossagent.

In another embodiment, the additional therapeutic agent is an H₃receptor antagonist/inverse agonist.

In still another embodiment, the additional therapeutic agent is anantidiabetic agent.

Cholesterol lowering agents useful in the combination therapies of thepresent invention, include but are not limited to: cholesterolbiosynthesis inhibitors; bile acid sequestrants; sterol absorptioninhibitors; 5-α-stanol absorption inhibitors; nicotinic acid and/ornicotinic acid receptor agonists; agonists or activators of peroxisomeproliferators-activated receptors (PPAR); ileal bile acid transport(“IBAT”) inhibitors (or apical sodium co-dependent bile acid transport(“ASBT”) inhibitors; nicotinic acid (niacin) and/or nicotinic acidreceptor agonists; acylCoA:cholesterol O-acyltransferase (“ACAT”)inhibitors; cholesteryl ester transfer protein (“CETP”) inhibitors;probucol or derivatives thereof, low-density lipoprotein (“LDL”)receptor activators; omega 3 fatty acids (“3-PUFA”); natural watersoluble fibers; plant sterols, plant stanols and/or fatty acid esters ofplant stanols.

Non-limiting examples of suitable bile acid sequestrants includecholestyramine (a styrene-divinylbenzene copolymer containing quaternaryammonium cationic groups capable of binding bile acids, such asQUESTRAN® or QUESTRAN LIGHT® cholestamine which are available fromBristol-Myers Squibb), colestipol (a copolymer of diethylenetriamine and1-chloro-2,3-epoxypropane, such as COLESTID® tablets which are availablefrom Pharmacia), colesevelam hydrochloride (such as WelChol® Tablets(poly(allylamine hydrochloride) cross-linked with epichlorohydrin andalkylated with 1-bromodecane and (6-bromohexyl)-trimethylammoniumbromide) which are available from Sankyo), water soluble derivativessuch as 3,3-ioene, N-(cycloalkyl) alkylamines and poliglusam, insolublequaternized polystyrenes, saponins and mixtures thereof. Suitableinorganic cholesterol sequestrants include bismuth salicylate plusmontmorillonite clay, aluminum hydroxide and calcium carbonate antacids.

Non-limiting examples of suitable cholesterol biosynthesis inhibitorsinclude inhibitors of HMG-CoA reductase, squalene synthase inhibitors,squalene epoxidase inhibitors and mixtures thereof.

Non-limiting examples of suitable HMG-CoA reductase inhibitors includestatins such as lovastatin (for example MEVACOR® which is available fromMerck & Co.), pravastatin (for example PRAVACHOL® which is availablefrom Bristol Meyers Squibb), fluvastatin, simvastatin (for exampleZOCOR® which is available from Merck & Co.), atorvastatin, cerivastatin,CI-981, resuvastatin, rivastatin and pitavastatin (such as NK-104 ofNegma Kowa of Japan), rosuvastatin; HMG-CoA reductase inhibitors, forexample L-659,699((E,E)-11-[3′R-(hydroxyl-methyl)-4′-oxo-2′R-oxetanyl]-3,5,7R-trimethyl-2,4-undecadienoicacid); squalene synthesis inhibitors, for example squalestatin 1; andsqualene epoxidase inhibitors, for example, NB-598((E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3′-bithiophen-5-yl)methoxy]benzene-methanaminehydrochloride) and other sterol biosynthesis inhibitors such as DMP-565.In one embodiment, an HMG-CoA reductase inhibitor is selected from thegroup consisting of lovastatin, pravastatin fluvastatin, simvastatin,atorvastatin, cerivastatin, pitavastatin, and rosuvastatin. In anotherembodiment, the HMG-CoA reductase inhibitor is lovastatin, pravastatinand simvastatin. In another embodiment, a suitable HMG-CoA reductaseinhibitor is simvastatin.

Weight loss agents useful in the combination therapies of the presentinvention include appetite suppressants, metabolic rate enhancers andnutrient absorption inhibitors. Appetite suppressant agents useful fortreating a Condition include cannabinoid receptor 1 (CB₁) antagonists orinverse agonists (e.g., rimonabant); Neuropeptide Y (NPY1, NPY2, NPY4and NTY5) antagonists; metabotropic glutamate subtype 5 receptor(mGluR5) antagonists (e.g., 2-methyl-6-(phenylethynyl)-pyridine and3[(2-methyl-1,4-thiazol-4-yl)ethynyl]pyridine); melanin-concentratinghormone receptor (MCH1R and MCH2R) antagonists; melanocortin receptoragonists (e.g, Melanotan-II and Mc4r agonists); serotonin uptakeinhibitors (e.g., dexfenfluramine and fluoxetine); serotonin (5HT)transport inhibitors (e.g., paroxetine, fluoxetine, fenfluramine,fluvoxamine, sertaline and imipramine); norepinephrine (NE) transporterinhibitors (e.g., desipramine, talsupram and nomifensine); glirelinantagonists; leptin or derivatives thereof, opioid antagonists (e.g.,nalmefene, 3-methoxynaltrexone, naloxone and nalterxone); orexinantagonists; bombesin receptor subtype 3 (BRS3) agonists;Cholecystokinin-A (CCK-A) agonists; ciliary neurotrophic factor (CNTF)or derivatives thereof (e.g., butabindide and axokine); monoaminereuptake inhibitors (e.g., sibutramine); glucagons-like peptide 1(GLP-1) agonists; topiramate; and phytopharm compound 57. Metabolic rateenhancers include acetyl-CoA carboxylase-2 (ACC2) inhibitors; betaadrenergic receptor 3 (β3) agonists; diacylglycerol acyltransferaseinhibitors (DGAT1 and DGAT2); fatty acid synthase (FAS) inhibitors(e.g., Cerulenin); phosphodiesterase (PDE) inhibitors (e.g.,theophylline, pentoxifylline, zaprinast, sildenafil, amrinone,milrinone, cilostamide, rolipram and cilomilast); thyroid hormone βagonists; uncoupling protein activators (UCP-1,2 or 3) (e.g., phytanicacid, 4-[(E)-2-(5,6,7,8-tetamethyl-2-naphtbalenyl)-1-propenyl]benzoicacid and retinoic acid); acyl-estrogens (e.g., oleoyl-estrone);glucocorticoid antagonists; 11-beta hydroxyl steroid dehydrogenase type1 (11β HSD-1) inhibitors; melanocortin-3 receptor (Mc3r) agonists; andstearoyl-CoA desaturase-1 (SCD-1) compounds. Nutrient absorptioninhibitors include lipase inhibitors (e.g., orlistat, lipstatin,tetrahydrolipstatin, teasaponin and diethylumbelliferyl phosphate);fatty acid transporter inhibitors; dicarboxylate transporter inhibitors;glucose transporter inhibitors; and phosphate transporter inhibitors.

Cholesterol absorption inhibitors useful in the combination therapies ofthe present invention include, but are not limited to, ezetimibe.

Other additional therapeutic agents useful in the combination therapiesof the present invention include, but are not limited to, rimonabant,phentermine, 2-methyl-6-(phenylethynyl)-pyridine,3[(2-methyl-1,4-thiazol-4-yl)ethynyl]pyridine, Melanotan-II,dexfenfluramine, fluoxetine, paroxetine, fenfluramine, fluvoxamine,sertaline, imipramine, desipramine, talsupram, nomifensine, leptin,nalmefene, 3-methoxynaltrexone, naloxone, nalterxone, butabindide,axokine, sibutramine, topiramate, phytopharm compound 57, Cerulenin,theophylline, pentoxifylline, zaprinast, sildenafil, amrinone,milrinone, cilostamnide, rolipram, cilomilast, phytanic acid,4-[(E)-2-(5,6,7,8-tetramnethyl-2-naphthalenyl)-1-propenyl]benzoic acid,retinoic acid, oleoyl-estrone, orlistat, lipstatin, tetrahydrolipstatin,teasaponin and diethylumbelliferyl phosphate.

Examples of antidiabetic agents useful in the present methods fortreating Type II diabetes include sulfonylureas, insulin sensitizers(such as PPAR agonists, DPPFV inhibitors, PTP-1B inhibitors andglucokinase activators), β-glucosidase inhibitors, insulinsecretagogues, hepatic glucose output lowering compounds, and insulin.

Non-limiting examples of useful sulfonylurea drugs include glipizide,tolbutamide, glyburide, glimepiride, chlorpropamide, acetohexamide,gliamilide, gliclazide, glibenclamide and tolazamide. Insulinsensitizers include PPAR-γ agonists described in detail above,preferably troglitazone, rosiglitazone, pioglitazone and englitazone;biguanidines such as metformin and phenformin; DPPIV inhibitors such assitagliptin, saxagliptin, denagliptin and vildagliptin; PTP-1Binhibitors; and glucokinase activators. β-Glucosidase inhibitors thatcan be useful in treating type II diabetes include miglitol, acarbose,and voglibose. Hepatic glucose output lowering drugs include Glucophageand Glucophage XR. Insulin secretagogues include sulfonylurea andnon-sulfonylurea drugs such as GLP-1, exendin, GIP, secretin, glipizide,chlorpropamide, nateglinide, meglitinide, glibenclamide, repaglinide andglimepiride. Insulin includes all formualtions of insulin, includinglong acting and short acting forms of insulin.

The Compound of Formula I may be administered in combination withweight-loss agents for the treatment of diabetes. Examples ofweight-loss agents useful in the present methods for treating diabetesinclude those listed above herein.

For treating diabetes, compounds of the invention may also beadministered in combination with antihypertensive agents, for exampleβ-blockers and calcium channel blockers (for example diltiazem,verapamil, nifedipine, amlopidine, and mybefradil), ACE inhibitors (forexample captopril, lisinopril, enalapril, spirapril, ceranopril,zefenopril, fosinopril, cilazopril, and quinapril), AT-1 receptorantagonists (for example losartan, irbesartan, and valsartan), renininhibitors and endothelin receptor antagonists (for examplesitaxsentan).

Certain meglitinide drugs lower blood glucose levels by stimulating therelease of insulin from the pancreas. This action is dependent uponfunctioning β cells in the pancreatic islets. Insulin release isglucose-dependent and diminishes at low glucose concentrations. Themeglitinide drugs close ATP-dependent potassium channels in the β cellmembrane by binding at characterizable sites. This potassium channelblockade depolarizes the β cell, which leads to an opening of calciumchannels. The resulting increased calcium influx induces insulinsecretion. Non-limiting examples of suitable meglitinide drugs useful inthe present methods include repaglinide and nateglinide.

Non-limiting examples of suitable antidiabetic agents that sensitize thebody to the insulin that is already present include certain biguanidesand certain glitazones or thiazolidinediones. Certain suitablebiguanides lower blood sugar by decreasing hepatic glucose production,decreasing intestinal absorption of glucose and improving insulinsensitivity (increasing peripheral glucose uptake and utilization). Anon-limiting example of a suitable biganide is metformin. Non-limitingexamples of metformin include metformin hydrochloride(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such asGLUCOPHAGE® Tablets from Bristol-Myers Squibb); metformin hydrochloridewith glyburide, such as GLUCOVANCE™ Tablets from Bristol-Myers Squibb);buformin.

Non-limiting examples of antidiabetic agents that slow or block thebreakdown of starches and certain sugars and are suitable for use in thecompositions of the present invention include alpha-glucosidaseinhibitors and certain peptides for increasing insulin production,Alpha-glucosidase inhibitors help the body to lower blood sugar bydelaying the digestion of ingested carbohydrates, thereby resulting in asmaller rise in blood glucose concentration following meals.Non-limiting examples of suitable alpha-glucosidase inhibitors includeacarbose; miglitol; camiglibose; certain polyamines as disclosed in WO01/47528 (incorporated herein by reference); voglibose. Non-limitingexamples of suitable peptides for increasing insulin productionincluding amlintide (CAS Reg. No. 122384-88-7 from Amylin; pramlintide,exendin, certain compounds having Glucagon-like peptide-1 (GLP-1)agonistic activity as disclosed in WO 00/07617 (incorporated herein byreference).

Non-limiting examples of additional antidiabetic agents include orallyadministrable insulin. Non-limiting examples of suitable orallyadministrable insulin or insulin containing compositions include AL-401from AutoImmune, and certain compositions as disclosed in U.S. Pat. Nos.4,579,730; 4,849,405; 4,963,526; 5,642,868; 5,763,396; 5,824,638;5,843,866; 6,153,632; 6,191,105; and International Publication No. WO85/05029 (each of which is incorporated herein by reference).

The present methods for treating NAFLD, include combination therapycomprising the administration of the Compound of Formula I and at leastone H₃ receptor antagonist/inverse agonist. H₃ receptorantagonists/inverse agonists are well-known in the art. H₃ receptorsites are found on sympathetic nerves, where they modulate sympatheticneurotransmission and attenuate a variety of end organ responses undercontrol of the sympathetic nervous system. Specifically, H₃ receptoractivation by histamine attenuates norepinephrine outflow to resistanceand capacitance vessels, causing vasodilation. H₃ receptorantagonists/inverse agonists are known to treat: allergy,allergy-induced airway (e.g., upper airway) responses, congestion (e.g.,nasal congestion), hypotension, cardiovascular disease, diseases of theGI tract, hyper and hypo motility and acidic secretion of thegastro-intestinal tract, obesity, sleeping disorders (e.g., hypersomnia,somnolence, and narcolepsy), disturbances of the central nervous system,attention deficit hyperactivity disorder (ADHD), hypo and hyperactivityof the central nervous system (for example, agitation and depression),and/or other CNS disorders (such as Alzheimer's, schizophrenia, andmigraine) in a patient such as a mammal. These compounds areparticularly useful for treating allergy, allergy-induced airwayresponses and/or congestion.

H₃ receptor antagonist/inverse agonists useful in the combinationtherapies of the present invention include, but are not limited to,imidazole type, such as those described in International PublicationNos. WO 95/14007 and WO 99/24405; non-imidazole H₃ receptor antagonistsdescribed in U.S. Pat. No. 6,720,328; indole derivatives described inU.S. Publication No. US 2004/0019099; benzimidazole derivativesdescribed in U.S. Publication No. US 2004/0048843A1 and U.S. PublicationNo. US 2004/0097483A1; and piperidine compounds described in U.S. Pat.No. 6,849,621. The above-listed patents and applications relating to H₃antagonists/inverse agonists are incorporated herein by reference.

In one embodiment, the invention provides a method for treating NAFLD ina patient, comprising administering: (i) an effective amount of aCompound of Formula I, (ii) an HMG-CoA reductase inhibitor, and (iii) abile acid sequestrant.

In another embodiment, the invention provides a method for treatingNAFLD in a patient, comprising administering: (i) an effective amount ofa Compound of Formula I, (ii) an HMG-CoA reductase inhibitor, and (iii)a weight loss agent.

In another embodiment, the invention provides a method for treatingNAFLD in a patient, comprising administering: (i) an effective amount ofa Compound of Formula I, (ii) an HMG-CoA reductase inhibitor, and (iii)a PPAR activator.

In still another embodiment, the invention provides a method fortreating NAFLD in a patient, comprising administering: (i) an effectiveamount of a Compound of Formula I, (ii) an HMG-CoA reductase inhibitor,and (iii) a sterol absorption inhibitor.

In yet another embodiment, the invention provides a method for treatingNAFLD in a patient, comprising administering: (i) an effective amount ofa Compound of Formula I, (ii) an HEMG-CoA reductase inhibitor, and (iii)a 5-α-stanol absorption inhibitor.

In a further embodiment, the invention provides a method for treatingNAFLD in a patient, comprising administering: (i) an effective amount ofa Compound of Formula I, (ii) an HMG-CoA reductase inhibitor, and (iii)a cholesterol absorption inhibitor.

In another embodiment, the invention provides a method for treatingNAFLD in a patient, comprising administering: (i) an effective amount ofa Compound of Formula I, (ii) an HMC-CoA reductase inhibitor, and (iii)a H₃ receptor antagonist/inverse agonist.

In one embodiment, the invention provides a method for treating obesityin a patient, comprising administering: (i) an effective amount of aCompound of Formula I, and (ii) a weight loss agent.

In another embodiment, the invention provides a method for treatingmetabolic syndrome in a patient, comprising administering. (i) aneffective amount of a Compound of Formula I, and (ii) a weight lossagent.

In another embodiment, the invention provides a method for treatingdiabetes in a patient, comprising administering: (i) an effective amountof a Compound of Formula I, and (ii) an antidiabetic agent.

In another embodiment, the invention provides a method for treatingdiabetes in a patient, comprising administering: (i) an effective amountof a Compound of Formula I and (ii) a weight loss agent.

Compositions and Administration

For preparing pharmaceutical compositions from the compound of thisinvention, inert, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,dispersible granules, capsules, cachets and suppositories. The powdersand tablets may be comprised of from about 5 to about 95 percent activeingredient. Suitable solid carriers are known in the art, e.g. magnesiumcarbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders,cachets and capsules can be used as solid dosage forms suitable for oraladministration. Examples of pharmaceutically acceptable carriers andmethods of manufacture for various compositions may be found in A.Gennaro (ed.), The Science and Practice of Pharmacy, 20^(th) Edition,(2000), Lippincott Wiliams & Wilkins, Baltimore, Md.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection or addition of sweeteners and opacifiers fororal solutions, suspensions and emulsions. Liquid form preparations mayalso include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas,e.g. nitrogen.

Also included are solid from preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

In one embodiment, the compound is administered orally.

In another embodiment, the pharmaceutical preparation is in a unitdosage form. In such form, the preparation is subdivided into suitablysized unit doses containing appropriate quantities of the activecomponent, e.g., an effective amount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 1 mg to about 150 mg. In one embodiment,quantity of active compound in a unit dose of preparation is from about1 mg to about 75 mg. In another embodiment, quantity of active compoundin a unit dose of preparation is from about about 1 mg to about 50 mg,according to the particular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage regimen for a particular situation iswithin the skill of the art. For convenience, the total daily dosage maybe divided and administered in portions during the day as required.

The amount and frequency of administration of the compounds of theinvention and/or the pharnaceutically acceptable salts thereof will beregulated according to the judgnent of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as seventy of the symptoms being treated. A typical recommendeddaily dosage regimen for oral administration can range from about 1mg/day to about 300 mg/day. In one embodiment, a daily oral dosage isfrom about 1 mg/day to 75 mg/day, in two to four divided doses.

Similarly, when the invention comprises a combination of the compound ofthis invention and another therapeutic agent for treating a Condition,the two active components may be co-administered simultaneously orsequentially, or a single pharmaceutical composition comprising thecompound of this invention and another compound in a pharmaceuticallyacceptable carrier can be administered. The components of thecombination can be administered individually or together in anyconventional dosage form such as capsule, tablet, powder, cachet,suspension, solution, suppository, nasal spray, etc. In one embodiment,the dose of the other therapeutic agent ranges from about 1 mg to about1000 mg per dose. The exact dose, however, is determined by publishedmaterial or by the attending clinician and is dependent on such factorsas the potency of the compound administered, the age, weight, conditionand response of the patient.

In various embodiments, non-limiting dosage ranges for selected othertherapeutic agents useful in the present methods are set forth below.The exact dose, however, is determined by published material or by theattending clinician and is dependent on such factors as the potency ofthe compound administered, the age, weight, condition and response ofthe patient.

In one embodiment, a total daily dosage of cholesterol biosynthesisinhibitor(s) can range from about 0.1 to about 160 mg per day. In oneembodiment, the dosage is from about 0.2 to about 80 mg/day,administered in a single dose or in 2-3 divided doses.

In another embodiment, a total daily dosage of peroxisomeproliferator-activated receptor(s) activator(s) can range from about 50to about 3000 mg per day. In one embodiment, the daily dose is fromabout 50 to about 2000 mg per day, administered in a single dose or in2-4 divided doses.

In another embodiment, a total daily dosage of IBAT inhibitor(s) canrange from about 0.01 to about 1000 mg/day. In one embodiment, thedosage is from about 0.1 to about 50 mg/day, administered in a singledose or in 2-4 divided doses.

In yet another embodiment, a total daily dosage of nicotinic acid canrange from about 500 to about 10,000 mg/day. In one embodiment, thedosage is from about 1000 to about 8000 mg/day. In another embodiment,the dosage is from about 3000 to about 6000 mg/day, administered in asingle dose or in divided doses. Generally, the total daily dosage of aNAR agonist can range from about 1 to about 100 mg/day.

In further embodiment, a total daily dosage of ACAT inhibitor(s) canrange from about 0.1 to about 1000 mg/day, administered in a single doseor in 2-4 divided doses.

In another embodiment, a total daily dosage of CETP inhibitor(s) canrange from about 0.01 to about 1000 mg/day, and preferably about 0.5 toabout 20 mg/kg/day, administered in a single dose or in 2 or moredivided doses.

In one embodiment, a total daily dosage of probucol or derivativesthereof can range from about 10 to about 2000 mg/day. In one embodiment,the dosage is from about 500 to about 1500 mg/day, administered in asingle dose or in 2-4 divided doses.

In another embodiment, a total daily dosage of LDL receptor activator(s)can range from about 1 to about 1000 mg/day, administered in a singledose or in 2-4 divided doses.

In another embodiment, a total daily dosage of fish oil or Omega 3 fattyacids can range from about 1 to about 30 grams per day, administered ina single dose or in 2-4 divided doses.

In still another embodiment, a total daily dosage of natural watersoluble fibers can range from about 0.1 to about 10 grams per day,adninistered in a single dose or in 2-4 divided doses.

In another embodiment, a total daily dosage of plant sterols, plantstanols and/or fatty acid esters of plant stanols can range from about0.5 to about 20 grams per day, administered in a single dose or in 2-4divided doses.

In a further embodiment, the total daily dosage of antidiabetic agentscan range from about 1 to about 3000 mg per day. In one embodiment, thetotal daily dose ranges from about 50 to about 2000 mg per day,administered in a single dose or in 2-4 divided doses.

When separate pharmaceutical compositions comprising a compound of thisinvention and another compound for treating a Condition are to beadministered, they can be provided in a kit comprising a single package,wherein the single package comprises a first container containing theCompound of Formula I and a pharmaceutically acceptable carrier, and oneor more additional separate containers, wherein each separate containercontains an additional therapeutic agent and a pharmaceuticallyacceptable carrier, with the compounds and agents being present inamounts such that the combination is therapeutically effective. A kit isadvantageous for administering a therapeutic combination when, forexample, the components of the therapeutic combination must beadministered at different time intervals or when they are in differentdosage forms.

EXAMPLES

General Methods

All solvents and reagents were used as received. Proton NMR spectra wereobtained using a Bruker AV 500 (500 MHz) instrument and were reported asparts per million (ppm) downfield from Me₄Si. LCMS analysis wasperformed using an Applied Biosystems API-100 mass spectrometer equippedwith a Shimadzu SCL-10A LC column: Altech platinum C18, 3 um, 33 mm×7 mmID; gradient flow: 0 min, 10% CH₃CN; 5 min, 95% CH₃CN; 7 min, 95% CH₃CN;7.5 min, 10% CH₃CN; 9 min, stop. Flash column chromatography wasperformed using Selecto Scientific flash silica gel, 32-63 mesh.Analytical and preparative TLC was performed using Analtech Silica gelGF plates. Chiral HPLC was performed using a Varian PrepStar systemequipped with a Chiralpak OD colunm (Chiral Technologies).

Example 1 Preparation of the Compound of Formula I

Step 1

LiAlH₄ (10.0 g, 0.264 mol, 1.24 eq) was added portionwise to a solutionof methyl-2-chloro-6-methylpyridine-4-carboxylate 1 (39.62 g, 0.213 mol)in dry Tf (800 mL) at room temperature with stirring over a period of1.4 h. The resulting mixture was stirred for 1 h and quenched withwater. 15% aqueous NaOH (100 mL) was added, followed by aqueoussodium-potassium tartrate (1 L). The resulting mixture was stirred for afurther 1.25 h and extracted with dichloromethane (2×1 L) to give, afterconcentration, (2-chloro-6-methylpyridin-4-yl)-methanol 2 (31.06 g, 93%)as a yellow solid.

Step 2

A bomb was charged with 2 (30.0 g, 0.190 mol) and aqueous conc NH₃ (225mL) and the resulting mixture was heated at 210° C. for 20 h. The systemwas cooled to room temperature, the volatiles removed in vacito and theresidue purified by column chromatography (dichloromethane: 0.4N NH₃ inMeOH 9:1) to give (2-aminio-6-methylpyridine-4-yl)-methanol as a mixtureof free base and hydrochloride salt. The mixture was redissolved indichloromethane:i-PrOH 1:1 (1 L) and treated with 20% aqueous NaOH (500mL). The layers were separated and the organic phase extracted withdichloromethane:i-PrOH 1:1 (1×1 L). The combined organic phase was driedand the solvent evaporated to give (2-amino-6-methylpyridine-4-yl)-methanol 3 (15.51 g, 59%) as pale orange crystals.

Step 3

Di-tert-butyl dicarbonate (105.75 g, 0.485 mol, 4.33 eq) was added to astirred solution of 3 (15.51 g, 0.112 mol) in tert-butyl alcohol (500mL) at room temperature. The resulting mixture was heated at 95° C. for19 h under a N₂ atmosphere, then cooled to room temperature and thesolvent evaporated in vacuo. The resulting brown oil was purified bycolumn chromatography (EtOAc:hexanes 1:1) to give the diprotectedaminoalcohol (38.25 g) as a yellow solid. 25% aqueous NaOH (150 mL) wasadded to a solution of the above material in MeOH (500 mL) over a periodof 10 min. The resulting mixture was stirred for 1 h, diluted with water(200 mL) and extracted with dichloromethane (2×750 mL) to give(4-hydroxymethyl-6-methyl-pyidine-2-yl)-carbamic acid tert-butyl ester 4(21.0 g, 79% over two steps) as an orange foam.

Step 4

Dess-Martin periodinane (50.0 g, 0.118 mol, 1.34 eq) was portlonwiseadded to a solution of 4 (21.0 g, 0.088 mol) in dichloromethanepyridine10:1 (1.1 L). The resulting solution was stirred at room temperature for2 h and then water (700 mL) was added. The mixture was stirred for afurther 5 min, and then the layers were separated. The aqueous layer wasextracted with dichloromethane (1×1 l), the combined organic phase driedand the solvent evaporated to give a brown solid which was purified bycolumn chromatography (EtOAc:hexane 1:2) to afford(4-formyl-6-methyl-pyridine-2-yl)-carbamic acid tert-butyl ester 5 (20.5g, 99%) as a pale orange solid.

Step 5

NaBH(OAc)₃ (57.8 g, 0.274 mol, 1.6 eq) was added to a solution ofpiperidine 6 (69.97 g, 0.171 mol, prepared using the method described inExample 2, below) and 5 (52.6 g, 0.223 mol, 1.3 eq in drydichloromethane (5.4 L) at room temperature with stirring. The resultingmixture was stirred for 20 h and then washed with aqueous K₂CO₃. Thelayers were separated and the aqueous layer extracted withdichloromethane (1×2 L). The combined organic phase was dried and thesolvent evaporated in vacuo to give an orange foam which was purified bycolumn chromatography (dichloromethane: 0.4 N NH₃ in MeOH 95:5) toafford 7 (93.38 g, 54%) as a yellow foam.

Step 6

TFA (900 mL) was added to a solution of 7 (93.38 g, 0.149 mol) indichloromethane (2.7 L). The resulting solution was stirred under a N₂atmosphere for 26 h, then cooled to 0° C. and carefully basified with15% aqueous ammonia solution. The layers were separated and the aqueouslayer extracted with dichloromethane (1×1.5 L). The combined organicphase was dried and the solvent removed in vacuo to give a pale yellowsolid which was triturated with dichloromethane (200 mL) and washed withdiethyl ether (200 mL) to afford the Compound of Formula I (59.05 g,75%) as a white solid. ¹H NMR (500 MHz, CDCl₃, δ=7.29): 1.96-2.16 (m,4H), 2.28-2.48 (m, 4H), 2.40 (s, 3H), 2.80 (m, 3H), 3.02-3.24 (m, 3H),3.42 (s, 2H), 4.37 (s, 2H), 4.70 (m, 1H), 4.83 (m, 1H), 5.84 (m, 1H),6.38 (s, 1H), 6.55 (s, 1H), 7.26 (dd, J=8.2, 4.7 Hz, 1H), 7.44 (ddd,J=7.5, 5.1, 1.2 Hz, 1H), 7.92 (app. td, J=<7.7>, 1.9 Hz, 1H), 8.09 (dd,J=8.0, 1.5 Hz, 1H), 8.32 (app. dt, J=8.0, <1.0> Hz, 1H), 8.40 (dd,J=4.7, 1.5 Hz), 8.74 (ddd, J=5.1, 1.9, 1.2 Hz, 1H). (C₂₉H₃₃FN₈O foundM+H 529.3).

The preparation of the compound of Formula I can be realized in manyways known to those skilled in the art. While this example provides oneparticular method for preparing this compound; other procedures, forexample those described in US 2004/0097483, are also applicable.

Example 2 Preparation of Intermediate Compound 6

Step 1:

A solution of compound 8 (100 g, 0.389 mol) in THF (400 mL) was addeddropwise over 1 h to a solution of lithium diisopropylamide (233 mL, 2.0M in THF/heptane/ethylbenzene, 0.466 mol) in THF (300 mL) at 0° C. Thered-orange solution was stirred at 0° C. for 30 min, and thentransferred by cannula to a pre-cooled (0° C.) solution ofN-fluorobenzenesulfonimide (153 g, 0.485 mol) in dry THF (600 mL). Thereaction mixture was stirred at 0° C. for 30 min, and then at 20° C. for18 h. The total solvent volume was reduced to approximately one third,and EtOAc (1 L) was added. The solution was washed successively withwater, 0.1 N aqueous HCl, saturated aqueous NaHCO₃, and brine. Theorganic layer was dried over MgSO₄, filtered, and concentrated underreduced pressure to yield a crude liquid. Separation by flashchromatography (hexanes-EtOAc 6:1) gave compound 9 (93.5 g, 87%).

Step 2:

A solution of 9 (50 g, 0.181 mol) in THF (300 mL) and MeOH (200 mL) wastreated with a solution of LiOH—H₂O (9.2 g, 0.218 mol) in water (100 mL)and then heated to 45° C. for 6 h. The mixture was then concentrated anddried in vacuo to provide 10 (45 g, 100%).

Step 3:

Compound 10 (20.4 g, 0.081 mol) was added slowly to a stirred flask ofdichloromethane (250 mL) at 20° C. The resulting white slurry was cooledto 0° C. and treated slowly with oxalyl chlorde (6.7 mL, 0.075 mol) anda drop of DMF. After stirring at 20° C. for 0.5 h, e mixture wasconcentrated and dried in vacuo to provide 11.

Step 4A:

A mixture of 12 (64 g, 0.40 mol), 13 (84 mL, 0.52 mol), and K₂CO₃ (66 g,0.48 mol) in anhydrous toluene (350 mL) was heated at reflux overnight.The reaction mixture was diluted with dichloromethane, washed threetimes with 5% aqueous NaOH, dried over Na₂SO₄, and concentrated.Recrystallization with MeOH provided 14 (121 g, 99%) as a yellow solid.

Step 4B:

A suspension of 14 (121 g, 0.41 mol) and Raney Nickel (10 g) in EtOH(400 mL) was shaken under H₂ (40 psi) for 4 h. The mixture was filteredthrough a short pad of celite (washing with MeOH). The filtrate wasconcentrated and dried in vacuo to provide 15 (109 g, 99%) as a darkbrown solid.

Step4C:

A solution of 15 (109 g, 0.41 mol) in dichloromethane:DMF 1:1 (500 mL)was treated with picolinic acid (61 g, 0.50 mol), EDCI (119 g, 0.62mol), HOBt (84 g, 0.62 mol) and diisopropylethylamine (141 mL, 1.03mol). The mixture was stirred at 70° C. for 6 h and then overnight at20° C. The reaction mixture was diluted with EtOAc, washed 3 times with5% aqueous NaOH, dried over Na₂SO₄, and concentrated. Flashchromatography (0-100% EtOAc/hexane) provided 16 (131 g, 86%).

Step 4D:

A solution of 16 (131 g, 0.36 mol) in acetic acid (200 mL) was heated at120° C. overnight. The reaction mixture was cooled, carefully basifiedwith 5% aqueous NaOH and extracted with CH₂Cl₂. The combined organicextracts were dried over Na₂SO₄ and concentrated. Flash chromatography(0-80% EtOAc/hexane) provided 17 (95 g, 76%) as a yellow solid.

Step 4E:

A solution of 17 (95 g, 0.27 mol) in anhydrous CHCl₃ (300 mL) wastreated with iodotrimethylsilane (272 g, 1.36 mol) and heated at 70° C.for 5 h. The reaction mixture was cooled, quenched with cold 10% aqueousNaOH, and extracted with dichloromethane. The combined organic extractswere dried over Na₂SO₄ and concentrated. Flash chromatography (2NNH₃-MeOH/EtOAc) provided 18 (43 g, 57%) as a pale yellow solid.

Step 5:

A mixture of 18 (0.075 mol) in dichioromethane (250 mL) was treated with11 (15 g, 0.054 mol) and diisopropylethylamine (25 mL, 0.135 mol) whilemaintaining a temperature of 20° C. After 1 h, the mixture wasconcentrated and then stirred in MeOH:dichloromethane:H₂O (200 mL:200mL:1 mL) for 1 h at 20° C. The solvent was then evaporated. Treatmentwith TFA (200 mL) in CH₂Cl₂ (250 mL) at 20° C. followed by flashchromatography (0-7% 7N NH₃—CH₃OH/CH₂Cl₂) provided 6 (80-90% from 10).

Example 3 Human H₃-Receptor Binding Assay

Binding assays were performed with membranes from HEK-293 cells stablyexpressing recombinant human histamine H₃ receptor. Each 200 μL assayvolume contained 1.0 nM [³H]N^(−α)-methylhistamine, test compound, and 3μg of membrane protein in 50 mM Tris-HCl, pH 7.4. Total binding wasdetermined in the absence of compound and nonspecific binding in thepresence of 10⁻⁵ M thioperamide. Material was incubated 30 minutes at30° C. then filtered on GF/B filters. These were rinsed three times withcold buffer, then dried, impregnated with Meltilex scintillationcounting medium, and counted. K_(i) values were determined from standardcurve-fitting procedures. For compounds generating less than 50%inhibition at the highest concentration tested, the percent inhibitionat this concentration was recorded. Values are averages from two assayswith duplicate determinations in each. Errors are expressed as the rangeof these values from the mean.

The compound of formula I has a K_(i) of 4.6 nM in the recombinant humanreceptor assay.

Example 4 In Vitro Binding of the Compound of Formula I to Canine andFeline H₃ Receptors

Binding assays were performed with membranes from a P2 pelletpreparation of brain from each species as described in West et al., MolPharmacol 38:610-613 (1990). Frozen brains were thawed at roomtemperature and then disrupted in ten volumes (w:v) of ice-cold 50 mMTris-HCl, pH 7.4, with a Polytron. Homogenates were centrifuged at1000×g and supernatants then centrifuged at 50,000×g. Pellets from thesecond centrifugation were resuspended in buffer, sedimented again at50,000×g, and stored frozen at −80° C.

Each 200 μL assay volume contained 1.0 nM [³H]N^(−α)-methylhlstamine,test compound, and 300 μg of membrane protein in 50 mM Tris-HCl, pH 7.4.Total binding was determined in the absence of compound and nonspecificbinding in the presence of 10⁻⁵ M thioperamide. Assay mixtures areincubated 30 minutes at 30° C. then filtered on CF/B filters. Filterswere rinsed three times with cold buffer, dried, impregnated withMeltilex scintillation counting medium, and counted. IC₅₀ values weredetermined using standard curve-fitting procedures and K_(i) values werecalculated from these using the method set forth in Cheng et al.,Biochem Pharmacol 22:3099-3108 (1973). Each competition binding assaycomprised ten concentrations of compound, each concentration assayed induplicate. Binding data obtained for the Compound of Formula I to canineand feline H₃ receptors are set forth below in Table 1. Ki values areaverages plus or minus the standard error of the mean from multipleassays as indicated.

TABLE 1 Binding of the Compound of Formula I to Canine and Feline H₃Receptors Species N K_(i) (nM) Dog 2 1.3 ± 0.2 Cat 3 4.9 ± 0.6

Example 5 Canine Pharmacokinetic Data for the Compound of Formula I

The Compound of Formula I was administered at a dose of 3 mg/kg tofasted beagle dogs orally (PO) at 3 mg/g (0.4% MC formulation) and i.v.at 3 mg/g (captisol, pH 5.1 fornulation). Blood samples were taken atmultiple time intervals for 48 hours post dosing. The blood samples wereconverted to plasma and stored at −20° C. until being assayed for theCompound of Formula I using an HPLC-API-MS/MS procedure. Thepharmacokinctic parameters that were calculated from thiis study aresumarized in Table 2 below. As shown in this table, the oralbioavailability of the Compound of Formula I in fasted dogs was 58%. Themean half-life after i.v. administration was 9.5 hr. The clearance was1.7 mL/min/kg.

TABLE 2 Pharmacokinetic Parameters of the Compound of the Invention inDogs after Oral (0.4% MC) and IV (captisol, pH 5.1) administration OralIV Parameter (units) (N = 3) (N = 3) Dose (mg/kg) 3 3 AUC (0-∞) 18.932.8 (μg · hr/mL) Bioavailability (%) 58 NA Half-life (hr) NA 9.5 MeanResidence Time (MRT) 18.6 10.9 (hr) Mean Absorption Time (MAT) 7.8 NA(MRT_(po) − MRT_(iv))(hr) Oral Cmax (μg/mL) 0.91 NA Clearance(mL/min/kg) NA 1.7 V_(d) (ss) (L/kg) NA 1.1 NA = not available

Example 6 Diet-Induced Obesity in Mice

Diet-induced obesity (DIO) was determined according to the followingprocedure. Young mice were maintained in individual cages at 22° C. on a12:12 hr light/dark cycle. Mice were made obese with a high fat diet.Mice were orally gavaged daily with vehicle or compound; body weight andfood intake were monitored daily.

The Compound of Formula I showed statistically significant inhibition ofweight gain in mice when administered orally once a day in 20% aqueoushydroxypropyl-β-cyclodextrin (HPBCD) at dose of 0.3 mg/kg. Compoundsspecifically disclosed in US 2004/0097483 typically have minimumefficacious dose of 3 mg/kg or higher.

Example 7 Effect of the Compound of Formula I on Liver Triglycerides inObese Mice

The Compound of Formula I was administered daily for 12 days via gavageto mice having diet-induced obesity at a dosage of 10 mg/kg/day (vehicle0.4% aqueous methylcellulose (MC); see procedure for diet-inducedobesity described above).

On day 12, the animals were sacrificed, their livers were collected, andliver pieces were extracted according to the procedure of Folch et al.,J. Biol. Chem. 226:497-509 (1957). The collected liver tissue was thenhomogenized in 6 mL of chloroform:methanol (2:1), 4 mL water was addedto the homogenized mixture and the resulting solution was vortexed, thencentrifuged at 1000×g for 30 minutes. The chloroform layer was removedand dried under nitrogen to provide the extracted lipids, which wereredissolved in 1 mL chloroform and aliquots were transferred into HPLCsample vials. Samples were dried under nitrogen and redissolved in 1 mLhexane:isopropanol (98.8:1.2).

Chromatography was performed using an isocratic mobile phase containing98.8% hexane and 1.2% isopropanol at a flow rate of 2 mL/min through aZorbax Sil (4.6×25 cm) silica column (Agilent Technologies #880952-701).Lipids in a 5 μL injection were detected by absorbance at 206 nm andquantitated by computer integration (System Gold, Beckman) of elutionprofiles. Cholesterol, cholesteryl ester, and triglycerideconcentrations were determined by comparison to standard curves usingNon-polar Lipid Mix-B, Matreya, Inc., Pleasant Gap, Pa. cat. #1130.

The results, illustrated in FIG. 1, show that the Compound of Formula Icaused statistically significant 52% reduction in liver triglycenrdecontent relative to control (average of 861±87 mg/liver in the compoundtreated group vs. 1789±135 mg/liver in the vehicle treated group).

Example 8 Assessment of Oral Tolerance of the Compound of Formula I inDogs

Male and female beagle dogs (N=6, age≧4 months, each having a bodyweight of from 10-15 kg) were used in this study. The treated animals(N=4) were orally administered the Compound of formula I once a day for7 days at a daily dosage of 5 mg/kg (5 times proposed efficacy dose).The control animals (N=2) were orally administered vehicle only. Animalswere fasted for 8 hours prior to each administration of the Compound offormula I or vehicle and were fasted for 2 hours after eachadministration of the Compound of formula I or vehicle. Blood sampleswere collected before, during, and after the treatment period forpharmacokinetic analysis.

Collection of Plasma Samples

Approximately 4.0±1 mL of blood was drawn from each animal at prior tothe study (day 0), at 24 hours after the first dose (day 1), and at theend of the study (day 7), using jugular venipuncture and immediatelyplaced into separated Vacutainer® tubes containing EDTA anticoagulant,and processed for plasma. The plasma was aspirated and divided into twoaliquots (≧0.3 ml each) and each aliquot was frozen at −70° C. or less.

Analysis of Plasma Samples

Collected blood plasma samples were analyzed for the concentration ofthe Compound of formula I using the following analytical method:

LC/MS Instrument:

Mass spectrometer: Finnegan (Thermoquest) Quantum

Ion source: APCI

Liquid Chromatograph: Shimadzu LC-10AD

Autosampler: LEAP Technologies HTS PAL

Computer. Gateway

Quantitative software: Xcalibur 2.0

Quantum Tuning Parameters:

Vaporizer temperature: 450° C.

Discharge current: 20v

Heated capillary temperature: 350° C.

Collision gas: Argon, 1.5 mTorr

Sheath gas pressure: Nitrogen, 49 psi

Aux. gas pressure: Nitrogen, 0 psi

MS/MS Scan Functions:

Compound Y

Parent Product Collision Energy Compound (m/z) (m/z) (eV) Compound offormula I 529.2 197 36 Compound Y (Internal Standard) 500.2 197 29

LC Conditions:

Mobile Phase:

-   -   A: 20/80% methanol:water, 10 mM ammonium acetate    -   B: 100% methanol    -   Flow Rate: 0.8 mL/min

Sample Processing Method:

A: Preparation of stock solution:

-   -   0.1, 1, 10, 100, 1000 ng/μL in 50:50 methanol:water (1000 in        DMSO) for standards.    -   1, 10, 100 ng/μL in 50:50 methanol:water for QCs.

B: Plasma standard curve and QC preparation: stock solution spiked inmatrix identical to samples.

-   -   Concentration of standard curve:        -   0, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000,            10000, 25000 ng/mL.    -   Concentration of QCs:        -   25, 250, 2500 ng/mL.

C: Internal Standard Solution: 0.1 ng/μL of Compound Y in acetonitrile.

D: Sample Preparation Procedure:

-   -   1) Pipette 40 μL of sample into a 1 mL 96-well plate.    -   2) Add 150 μL of internal standard solution to each well.    -   3) Gently vortex plate for 1 minute.    -   4) Centrifuge samples for 10 minutes (Eppendorf 5810 Centifuge).    -   5) Pipette 100 μL of supernatant into a 350 μL 96-well plate.        Results

Results of the above-described study are set forth below in Table 3:

TABLE 3 Blood Plasma Levels of the Compound of Formula I in TreatedAnimals Plasma level at Plasma level at Plasma level at Sex of animalDay 0 (ng/mL) Day 1 (ng/mL) Day 7 (ng/mL) male 0 118 263 male 6.8 157207 female 0 604 680 female 0 148 540 Mean 1.70 256.75 422.50

These results indicate that the Compound of formula I was accumulated 7days after daily oral administration of this compound. Accumulation wasmore pronounced in female dogs compared to male dogs. The compound waswell tolerated when administered daily at a dose of 5 mg/kg. No adverseevents were observed during the study.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in therelevant art and are intended to fall within the scope of the appendedclaims.

A number of references have been cited, the entire disclosures of whichhave been incorporated herein in their entirety.

1. A method of treating obesity comprising administering to a patient inneed of such treatment an effective amount of a compound having thestructure:


2. The method of claim 1, wherein the patient is a dog or a cat.